Effect of the tyrosine 96 hydrogen bond on the inactivation of cytochrome P-450cam induced by hydrostatic pressure

The effects of removal of the tyrosine 96 hydrogen bond on the stability and conformational events of cytochrome P-450cam are presented in this communication. Hydrostatic pressure has been used as a tool to perturbe the structure leading to the formation of cytochrome P-420, an inactivated but soluble and undenatured form of the enzyme. We show that the spin transition of cytochrome P-450cam, which is known to be influenced by hydrostatic pressure, is affected by this single mutation. The free energy of stabilisation of native substrate-free cytochrome P-450cam is not affected by the removal of the tyrosine 96 hydrogen bond via mutagenesis to phenylalanine, whereas the substrate-bound protein shows a difference of 21 kJ/mol. These results, as well as an observed 110 ml/mol difference for the volume of the inactivation reaction between substrate-bound native and mutant proteins, have been interpreted in terms of a more hydrated heme pocket for the site-directed mutant at position 96 compared to the wild-type protein where camphor is tightly bound via the tyrosine 96 hydrogen bond and water excluded from the active site.     

Effect of alcohols on binding of camphor to cytochrome P450cam: spectroscopic and stopped flow transient kinetic studies

Addition of alcohols to cytochrome P450cam (CYP101) was shown to release the substrate camphor from the heme pocket of the enzyme. The release of the substrate was found to be caused both due to increased solubility of the substrate in solution in presence of alcohol and due to change in the tertiary structure of the active site of the enzyme. The far-UV CD and near-UV CD spectra reveal that addition of alcohols to cytochrome P450cam cause a small change in the secondary structural elements but a significant change in the tertiary structural organization of this enzyme. The CD spectra at the heme region at various concentrations of alcohols indicate a substantial change in the tertiary structural organization around the heme moiety too. The equilibrium constant associated with the binding of camphor to Cyt P450cam is strongly dependent on the concentration of alcohols and the corresponding free energy associated with the binding is found to scale linearly with the concentration of alcohols. Kinetic experiments on binding of camphor to Cyt P450cam show that both k(on) and k(off) rate constants are strongly affected by addition of alcohols suggesting that alcohol expel camphor out of the heme cavity of Cyt P450cam by affecting tertiary structure of Cyt P450cam as well as by modifying the solubility properties of camphor in aqueous medium.     

Role of substrate on the conformational stability of the heme active site of cytochrome P450cam: effect of temperature and low concentrations of denaturants

The effect of 1R-camphor on the conformational stability of the heme active site of cytochrome P450cam has been investigated. The absorption spectra of the heme moiety showed the presence of two hitherto unknown intermediates formed at low urea concentrations or during small temperature perturbations. The corresponding thermodynamic parameters were obtained by global fitting of the experimental data to a generalized sequential unfolding model at different wavelengths, which showed that the active conformation of the enzyme is stabilized by binding of the substrate at the active site. Circular-dichroism spectra of the enzyme in the visible- and far-UV region were studied to identify the critical range of denaturant concentration and the temperature at which the tertiary structure around the heme center was affected with almost no change in the secondary structure of the enzyme. This critical range of urea concentration was 0–2.8 M in the presence of camphor and 0–1.5 M in the absence of camphor. The tertiary structure of the enzyme was found to undergo conformational change in the temperature range 20–60 °C in the presence of the substrate and 20–47 °C in its absence. The spectral assignments of the intermediate species of the heme active site with the intact secondary structure of the enzyme were made by deconvolution of the Soret absorption spectra, and the results were analyzed to determine stabilization of the heme active-site geometry by 1R-camphor. Results showed that subtle conformational changes due to melting of the tertiary contacts in the active site lead to formation of intermediates which are coordinatively similar to the native enzyme. Analogous intermediate species might be responsible for leakage in the redox catalytic cycle of the enzyme.     

Alteration of P450 distal pocket solvent leads to impaired proton delivery and changes in heme geometry

Distal pocket water molecules have been widely implicated in the delivery of protons required in O-O bond heterolysis in the P450 reaction cycle. Targeted dehydration of the cytochrome P450cam (CYP101) distal pocket through mutagenesis of a distal pocket glycine to either valine or threonine results in the alteration of spin state equilibria, and has dramatic consequences on the catalytic rate, coupling efficiency, and kinetic solvent isotope effect parameters, highlighting an important role of the active-site hydration level on P450 catalysis. Cryoradiolysis of the mutant CYP101 oxyferrous complexes further indicates a specific perturbation of proton-transfer events required for the transformation of ferric-peroxo to ferric-hydroperoxo states. Finally, crystallography of the 248Val and 248Thr mutants in both the ferric camphor bound resting state and ferric-cyano adducts shows both the alteration of hydrogen-bonding networks and the alteration of heme geometry parameters. Taken together, these results indicate that the distal pocket microenvironment governs the transformation of reactive heme-oxygen intermediates in P450 cytochromes.     

Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: a proposed molecular model for a cytochrome P-450cam electron-transfer complex

Cytochrome b5 has been genetically engineered to afford a fluorescent derivative capable of monitoring its association with cytochrome P-450cam from Pseudomonas putida [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. In the mutant cytochrome b5, threonine is replaced by a cysteine at position 65 (T65C) and has been labeled with the environmentally sensitive fluorophore acrylodan. In this paper, the physiological P-450cam reductant putidaredoxin, an Fe2S2.Cys4 iron-sulfur protein, is shown to competitively inhibit the cytochrome b5 association, suggesting that cytochrome b5 and putidaredoxin bind to a similar site on the cytochrome P-450cam surface. Since the crystal structures for both cytochrome b5 and cytochrome P-450cam have been solved to high resolution, the complex has been computer modeled, and a good fit was found on the proximal surface of nearest approach to the P-450cam heme prosthetic group. The proposed model includes electrostatic contacts between conserved cytochrome b5 carboxylates Glu-44, Glu-48, Asp-60, and the exposed heme propionate with cytochrome P-450cam basic residues Lys-344, Arg-72, Arg-112, and Arg-364, respectively. Putidaredoxin has similarly been shown to contain a carboxylate-based binding surface, and the current results suggest that if the model is correct, then it also interacts at the proposed site, probably utilizing similar P-450cam electrostatic contacts.     

Phe393 mutants of cytochrome P450 BM3 with modified heme redox potentials have altered heme vinyl and propionate conformations

It has been well established that the heme redox potential is affected by many different factors. Among others, it is sensitive to the proximal heme ligand and the conformation of the propionate and vinyl groups. In the cytochrome P450 BM3 heme domain, substitution of the highly conserved phenylalanine 393 results in a dramatic change in the heme redox potential [Ost, T. W. B., Miles, C. S., Munro, A. W., Murdoch, J., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 13421-13429]. We have used resonance Raman spectroscopy to characterize heme structural changes and modification of heme interactions with the protein matrix that are induced by the F393 substitutions and to determine their correlation with the heme redox potential. Our results show that the Fe-S stretching frequency of the 5-coordinated, high-spin ferric heme is not affected by the mutations, suggesting that the electron density in the Fe-S bond in this state is not affected by the F393 mutation and is not a good indicator of the heme redox potential. Substrate binding perturbs the hydrogen bonding between one propionate group and the protein matrix and correlates to both the size of residue 393 and the heme redox potential. However, heme reduction does not affect the conformation of the propionate groups. Although the conformation of the vinyl groups is not affected much by substrate binding, their conformation changes from mainly out-of-plane to predominantly in-plane upon heme reduction. The extent of these conformational changes correlates strongly with the size of the 393 residue and the heme redox potential, suggesting that steric interaction between this residue and the vinyl groups may be of importance in regulating the heme redox potential in the P450 BM3 heme domain. Further implications of our findings for the change in redox potential upon mutation of F393 will be discussed.     

Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam.

To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450cam, we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450cam from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm-1, indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate-binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O-O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450cam is important for the construction of the heme pocket and the heterolysis of the O-O bond.     

Determination of cytochrome b5 association reactions. Characterization of metmyoglobin and cytochrome P-450cam binding to genetically engineered cytochromeb5

Genetically engineered cytochrome b5 has been used to quantitative binding interactions of this protein with cytochrome P-450cam and sperm whale metmyoglobin by static fluorescence titration. Two cytochrome b5 mutants were constructed by cassette mutagenesis to replace a surface threonine residue with cysteine at two crystallographically defined positions, 65 and 8, located 11 and 21 A, respectively, from the nearest heme edge. The T65C and T8C mutant proteins were labeled with the sulfhydryl selective fluorescent reagent, acrylodan, which provided a spectral probe for monitoring protein-protein association. The fluorescence emission spectra of the acrylodan-labeled T65C mutant exhibited an ionic strength-dependent, blue-shifted fluorescence enhancement upon binding met-myoglobin, cytochrome c, and cytochrome P-450cam, whereas the acrylodan-labeled T8C mutant fluorescence emission remained unchanged during all titrations. Dissociation constants of 1.3, 0.6, and 0.5 microM, pH 7.15, were measured for metmyoglobin, cytochrome P-450cam, and cytochrome c, respectively. A similar averaged binding surface for cytochrome P-450cam and cytochrome c is suggested by their closely related degree of fluorescence enhancement, degree of emission blue shift, and binding free energies. Myoglobin binds less tightly, enhances fluorescence to a greater extent, and exhibits a larger blue shift in acrylodan emission spectra suggesting a different averaged binding orientation relative to the acrylodan probe.     

The roles of active site hydrogen bonding in cytochrome P-450cam as revealed by site-directed mutagenesis

The role of the active site hydrogen bond of cytochrome P-450cam has been studied utilizing a combination of site-directed mutagenesis and substrate analogues with altered hydrogen bonding capabilities. Cytochrome P-450cam normally catalyzes the regiospecific hydroxylation of the monoterpene camphor. The x-ray crystal structure of this soluble bacterial cytochrome P-450 (Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122-16128) indicates a specific hydrogen bond between tyrosine 96 and the carbonyl moiety of the camphor substrate. The site-directed mutant in which tyrosine 96 has been changed to a phenylalanine and the substrate analogues thiocamphor and camphane have been used to probe this interaction in several aspects of catalysis. At room temperature, both the mutant enzyme with camphor and the wild type enzyme with thiocamphor bound result in 59 and 65% high-spin ferric enzyme as compared to the 95% high spin population obtained with native enzyme and camphor as substrate. The equilibrium dissociation constant is moderately increased, from 1.6 microM for the wild type protein to 3.0 and 3.3 microM for wild type-thiocamphor and mutant-camphor complexes, respectively. Camphane bound to cytochrome P-450cam exhibits a larger decrease in high spin fraction (45%) and a correspondingly larger KD (46 microM), suggesting that the carbonyl moiety of camphor plays an important steric role in addition to its interaction as a hydrogen bond acceptor. The absolute regioselectivity of the mutant enzyme, and of the wild type enzyme with thiocamphor, is lost resulting in production of several hydroxylated products in addition to the 5-exo-hydroxy isomer. Based on rates of NADH oxidation, comparison of the substrate specificity for these systems (kcat/KD) indicates a 5- and 7-fold decrease in specificity for the mutant enzyme and thiocamphor-wild type complex, respectively. The replacement of the cytochrome P-450cam active site tyrosine with phenylalanine does not affect the branching ratio of monooxygenase versus oxidase chemistry or peroxygenase activity (Atkins, W.M., and Sligar, S.G. (1987) J. Am. Chem. Soc. 109, 3754-3760).     

Reversible inactivation of cytochrome P450 by alkaline earth metal ions: auxiliary metal ion induced conformation change and formation of inactive P420 species in CYP101

The effects of the divalent alkaline-earth metal ions (Ca²⁺ and Mg²⁺) on the substrate binding affinity, spin-state transition at the heme active site, conformational properties as well as the stability of the active form of cytochrome P450cam (CYP 101) have been investigated using various spectroscopic and kinetic methods. The divalent cations were found to have two types of effects on the enzyme. At the initial stage the alkaline-earth metal ion facilitated enhanced binding of the substrate and formation of the high-spin form of the heme active center of the enzyme compared to that in absence of any metal ion. However, analogous to many other mono-valent metal ions, the alkaline-earth metal ions were also less efficient than K⁺ in promoting the substrate binding and spin-transition properties of the enzyme. The auxiliary metal ions were shown to cause small but distinct change in the circular dichroism spectra of the substrate-free enzyme in the visible region, indicating that the tertiary structure around the heme was perturbed on binding of the auxiliary metal ion to the enzyme. The effect of the auxiliary metal ion was found to be more prominent in the WT enzyme compared to the Y96F mutant of P450cam suggesting that the Tyr 96 residue plays an important role in mediating the effects of the auxiliary metal ions to the active site of the enzyme. At the second stage of interaction, the alkaline-earth metal ions were found to slowly convert the enzyme into an inactive P420 form, which could be reversibly re-activated by addition of KCl. The results have been discussed in the light of understanding the mechanism of inactivation of certain mammalian P450 enzymes by these alkaline-earth metal ions.     

The role of serine-246 in cytochrome P450eryF-catalyzed hydroxylation of 6-deoxyerythronolide B

A strongly conserved threonine residue in the I-helix of cytochrome P450 enzymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronolide B hydroxylase (P450eryF) is unusual in that the conserved threonine residue is replaced by alanine in this enzyme. On the basis of the crystal structures of substrate-bound P450eryF, it has been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules 519 and 564, respectively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a threonine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were constructed and characterized. In each case, decreased catalytic activity and increased uncoupling could be correlated with changes in the hydrogen bonding environment. These results suggest that Ser-246 does indeed indirectly participate in the proton shuttling pathway, and also strongly support our previous hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction.     

The Role of Serine-246 in Cytochrome P450eryF-Catalyzed Hydroxylation of 6-Deoxyerythronolide B

A strongly conserved threonine residue in the I-helix of cytochrome P450 enzymes participates in a proton delivery system for binding and cleavage of dioxygen molecules. 6-Deoxyerythronol ide B hydroxylase (P450eryF) is unusual in that the conserved threonine residue is replaced by alanine in this enzyme. On the basis of crystal structures of substrate-bound P450eryF, it has been proposed that the C-5 hydroxyl group of the substrate and serine-246 of the enzyme form hydrogen bonds with water molecules 519 and 564, respectively. This hydrogen bonding network constitutes the proton delivery system whereby P450eryF maintains its catalytic activity in the absence of a threonine hydroxyl group in the conserved position. To further assess the role in the proton delivery system of hydroxyl groups around the active site, three mutant forms of P450eryF (A245S, S246A, and A245S/S246A) were constructed and characterized. In each case, decreased catalytic activity and increased uncoupling could be correlated with changes in the hydrogen bonding environment. These results suggest that Ser-246 does indeed participate in the proton shuttling pathway, and also support our previous hypothesis that the C-5 hydroxyl group of the substrate participates in the acid-catalyzed dioxygen bond cleavage reaction. Copyright 2000 Academic Press.     

Solution conformation of the His-47 to Ala-47 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551

In the cytochrome c-551 family, the heme 17-propionate caboxylate group is always hydrogen bonded to an invariant Trp-56 and conserved residues (His and Arg mainly, Lys occasionally) at position 47. The mutation of His-47 to Ala-47 for Pseudomas stutzeri ZoBell cytochrome c-551 removes this otherwise invariant hydrogen bond. The solution structure of ferrous H47A has been solved based on NMR-derived constraints. Results indicate that the mutant has very similar main chain folding compared to wild-type. However, less efficient packing of residues in the mutant surrounding the heme propionates leads to more solvent exposure for both propionate groups, which may account for decreased stability of the mutant. The mutant has a reduction potential different from wild-type, and furthermore, the pH dependence of this potential is not the same as for wild-type. The structure of the mutant suggests that these changes are related to the loss of the residue-47 propionate hydrogen bond and the loss of charge on the side chain of residue 47.     

Crystal structure of P450cin in a complex with its substrate, 1,8-cineole, a close structural homologue to D-camphor, the substrate for P450cam

Cytochrome P450cin catalyzes the monooxygenation of 1,8-cineole, which is structurally very similar to d-camphor, the substrate for the most thoroughly investigated cytochrome P450, cytochrome P450cam. Both 1,8-cineole and d-camphor are C(10) monoterpenes containing a single oxygen atom with very similar molecular volumes. The cytochrome P450cin-substrate complex crystal structure has been solved to 1.7 A resolution and compared with that of cytochrome P450cam. Despite the similarity in substrates, the active site of cytochrome P450cin is substantially different from that of cytochrome P450cam in that the B' helix, essential for substrate binding in many cytochrome P450s including cytochrome P450cam, is replaced by an ordered loop that results in substantial changes in active site topography. In addition, cytochrome P450cin does not have the conserved threonine, Thr252 in cytochrome P450cam, which is generally considered as an integral part of the proton shuttle machinery required for oxygen activation. Instead, the analogous residue in cytochrome P450cin is Asn242, which provides the only direct protein H-bonding interaction with the substrate. Cytochrome P450cin uses a flavodoxin-like redox partner to reduce the heme iron rather than the more traditional ferredoxin-like Fe(2)S(2) redox partner used by cytochrome P450cam and many other bacterial P450s. It thus might be expected that the redox partner docking site of cytochrome P450cin would resemble that of cytochrome P450BM3, which also uses a flavodoxin-like redox partner. Nevertheless, the putative docking site topography more closely resembles cytochrome P450cam than cytochrome P450BM3.     

Roles of residues 129 and 209 in the alteration by cytochrome b5 of hydroxylase activities in mouse 2A P450S.

Cytochrome b5 stimulates the coumarin 7-hydroxylation activity of P450coh. A mutation of Arg-129 in P450coh, however, abolishes the stimulation. Moreover, this mutant P450coh binds loosely to cytochrome b5-conjugated Sepharose 4B, whereas wild-type P450coh binds tightly. Consistent with this, the mutation increases the Ka value for b5 binding approximately 6-fold. The identity of residue 209 also alters the stimulation of the activity of P450coh depending on the type of the substrates used and products formed. Coumarin 7-hydroxylation activity is greatly stimulated by cytochrome b5 only when Phe is at position 209, while cytochrome b5 stimulates testosterone hydroxylation activity of P450coh in which Phe, Asn, Ser or Lys substitutes residue 209. P450coh changes its rate of hydrogen peroxide formation depending on the identity of residue 209 and substrate used. Cytochrome b5 decreases the hydrogen peroxide formation of some P450coh whose activities are stimulated by the cytochrome; however, the decrease does not always result in stimulating the activity. The results indicate, therefore, that residues 129 and 209 play different roles in stimulating P450coh activity by cytochrome b5; Arg-129 is a key residue in the cytochrome b5-binding domain and is essential for the stimulation. Residue 209, however, alters the efficiency of electron transport for substrate oxidation as a residue which resides near the sixth ligand of heme and in the substrate-binding site.     

Putidaredoxin-cytochrome p450cam interaction. Spin state of the heme iron modulates putidaredoxin structure

During the monooxygenase reaction catalyzed by cytochrome P450cam (P450cam), a ternary complex of P450cam, reduced putidaredoxin, and d-camphor is formed as an obligatory reaction intermediate. When ligands such as CO, NO, and O2 bind to the heme iron of P450cam in the intermediate complex, the EPR spectrum of reduced putidaredoxin with a characteristic signal at 346 millitesla at 77 K changed into a spectrum having a new signal at 348 millitesla. The experiment with O2 was carried out by employing a mutant P450cam with Asp251 --> Asn or Gly where the rate of electron transfer from putidaredoxin to oxyferrous P450cam is considerably reduced. Such a ligand-induced EPR spectral change of putidaredoxin was also shown in situ in Pseudomonas putida. Mutations introduced into the neighborhood of the iron-sulfur cluster of putidaredoxin revealed that a Ser44 --> Gly mutation mimicked the ligand-induced spectral change of putidaredoxin. Arg109 and Arg112, which are in the putative putidaredoxin binding site of P450cam, were essential for the spectral changes of putidaredoxin in the complex. These results indicate that a change in the P450cam active site that is the consequence of an altered spin state is transmitted to putidaredoxin within the ternary complex and produces a conformational change of the 2Fe-2S active center.     

Mutations in the putative H-channel in the cytochrome c oxidase from Rhodobacter sphaeroides show that this channel is not important for proton conduction but reveal modulation of the properties of heme a

As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase catalyzes the reduction of oxygen to water, concomitantly generating a proton gradient. X-ray structures of two cytochrome c oxidases have been reported, and in each structure three possible pathways for proton translocation are indicated: the D-, K-, and H-channels. The putative H-channel is most clearly delineated in the bovine heart oxidase and has been proposed to be functionally important for the translocation of pumped protons in the mammalian oxidase [Yoshikawa et al. (1998) Science 280, 1723-1729]. In the present work, the functional importance of residues lining the putative H-channel in the oxidase from Rhodobacter sphaeroides are examined by site-directed mutagenesis. Mutants were generated in eight different sites and the enzymes have been purified and characterized. The results suggest that the H-channel is not functionally important in the prokaryotic oxidase, in agreement with the conclusion from previous work with the oxidase from Paracoccus denitrificans [Pfitzner et al. (1998) J. Biomembr. Bioenerg. 30, 89-93]. Each of the mutants in R. sphaeroides, with an exception at only one position, is enzymatically active and pumps protons in reconstituted proteoliposomes. This includes H456A, where in the P. denitrificans oxidase a leucine residue substituted for the corresponding residue resulted in inactive enzyme. The only mutations that result in completely inactive enzyme in the set examined in the R. sphaeroides oxidase are in R52, a residue that, along with Q471, appears to be hydrogen-bonded to the formyl group of heme a in the X-ray structures. To characterize the interactions between this residue and the heme group, resonance Raman spectra of the R52 mutants were obtained. The frequency of the heme a formyl stretching mode in the R52A mutant is characteristic of that seen in non-hydrogen-bonded model heme a complexes. Thus the data confirm the presence of hydrogen bonding between the heme a formyl group and the R52 side chain, as suggested from crystallographic data. In the R52K mutant, this hydrogen bonding is maintained by the lysine residue, and this mutant enzyme retains near wild-type activity. The heme a formyl frequency is also affected by mutation of Q471, confirming the X-ray models that show this residue also has hydrogen-bonding interactions with the formyl group. Unlike R52, however, Q471 does not appear to be critical for the enzyme function.     

Engineering cytochrome c peroxidase into cytochrome P450: a proximal effect on heme-thiolate ligation.

In an effort to investigate factors required to stabilize heme-thiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered. The UV-visible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein. The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation. Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid [Choudhury et al. (1994) J. Biol. Chem. 267, 25656-25659]. Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable heme-thiolate ligation in the resting state of the enzyme. To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared. Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam. The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes. This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metal-ligand ligation.     

Hydrogen bond network of cytochrome P-450cam: a network connecting the heme group with helix K.

During investigations of the structural character of a mutant P-450cam where Glu-286 is replaced with lysine, we obtained evidence of a hydrogen bond network between helix K and the heme group via helix L of P-450cam. This mutant protein loses the ability to maintain the heme group in a proper position, possibly due to a break in the hydrogen bond network.     

Structural evidence for direct hydride transfer from NADH to cytochrome P450nor

Nitric oxide reductase cytochrome P450nor catalyzes an unusual reaction, direct electron transfer from NAD(P)H to bound heme. Here, we succeeded in determining the crystal structure of P450nor in a complex with an NADH analogue, nicotinic acid adenine dinucleotide, which provides conclusive evidence for the mechanism of the unprecedented electron transfer. Comparison of the structure with those of dinucleotide-free forms revealed a global conformational change accompanied by intriguing local movements caused by the binding of the pyridine nucleotide. Arg64 and Arg174 fix the pyrophosphate moiety upon the dinucleotide binding. Stereo-selective hydride transfer from NADH to NO-bound heme was suggested from the structure, the nicotinic acid ring being fixed near the heme by the conserved Thr residue in the I-helix and the upward-shifted propionate side-chain of the heme. A proton channel near the NADH channel is formed upon the dinucleotide binding, which should direct continuous transfer of the hydride and proton. A salt-bridge network (Glu71-Arg64-Asp88) was shown to be crucial for a high catalytic turnover.